Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Abstract

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

Small Molecule Screening Strategies from Lead Identification to Validation

“…Thus, four articles in this collection report on advances in this space. Festa et al describe a screening platform for kinase inhibitor discovery that uses self-assembled nucleic acid programmable protein arrays (NAPPA) 4 . These arrays display full-length human kinases and allow for multiplexed screening of compounds across a large kinase collection, thus allowing not only for identifying hits but for profiling selectivity as well.…”